U.S. patent number 10,620,419 [Application Number 14/904,062] was granted by the patent office on 2020-04-14 for arrangement for light sheet microscopy.
This patent grant is currently assigned to Carl Zeiss Microscopy GmbH. The grantee listed for this patent is Carl Zeiss Microscopy GmbH. Invention is credited to Thomas Kalkbrenner, Helmut Lippert, Jorg Siebenmorgen.
United States Patent |
10,620,419 |
Siebenmorgen , et
al. |
April 14, 2020 |
Arrangement for light sheet microscopy
Abstract
An arrangement for light sheet microscopy including: a sample
vessel, for receiving a medium containing sample, having a covering
and being oriented with respect to a planar reference surface;
illumination optics with an illumination objective for illuminating
the sample with a light sheet; and detection optics with a
detection objective. The optical axis of the illumination objective
and the light sheet lies in a plane that forms a nonzero
illumination angle with the normal of the reference surface. The
optical axis of the detection objective forms a nonzero detection
angle with the normal of the reference surface. A bulge is formed
at the covering for receiving the sample. The bulge has inner and
outer interfaces. The optical axes of the illumination objective
and detection objective form a minimal angle with the normals of
the interfaces at least in the region where the optical axes pass
through the interfaces.
Inventors: |
Siebenmorgen; Jorg (Jena,
DE), Kalkbrenner; Thomas (Jena, DE),
Lippert; Helmut (Jena, DE) |
Applicant: |
Name |
City |
State |
Country |
Type |
Carl Zeiss Microscopy GmbH |
Jena |
N/A |
DE |
|
|
Assignee: |
Carl Zeiss Microscopy GmbH
(Jena, DE)
|
Family
ID: |
51177058 |
Appl.
No.: |
14/904,062 |
Filed: |
July 8, 2014 |
PCT
Filed: |
July 08, 2014 |
PCT No.: |
PCT/EP2014/064550 |
371(c)(1),(2),(4) Date: |
January 08, 2016 |
PCT
Pub. No.: |
WO2015/004107 |
PCT
Pub. Date: |
January 15, 2015 |
Prior Publication Data
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|
|
Document
Identifier |
Publication Date |
|
US 20160170195 A1 |
Jun 16, 2016 |
|
Foreign Application Priority Data
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|
|
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Jul 10, 2013 [DE] |
|
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10 2013 107 298 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G02B
21/26 (20130101); G02B 21/367 (20130101); G02B
21/06 (20130101); A01K 13/004 (20130101); G02B
21/0076 (20130101); G02B 21/34 (20130101); G02B
27/0068 (20130101); G01N 21/6458 (20130101); G02B
21/16 (20130101); G02B 21/0032 (20130101) |
Current International
Class: |
G02B
21/34 (20060101); G02B 21/26 (20060101); G02B
27/00 (20060101); G02B 21/00 (20060101); G02B
21/06 (20060101); G02B 21/36 (20060101); G02B
21/16 (20060101) |
Field of
Search: |
;359/368,385,398,370,374
;356/244,246,318,450,904 ;382/128 ;422/68.1 ;436/63,172,809
;250/458.1,459.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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102 57 423 |
|
Jun 2004 |
|
DE |
|
10 2007 015 061 |
|
Oct 2008 |
|
DE |
|
10 2007 015061 |
|
Oct 2008 |
|
DE |
|
10 2007 048 409 |
|
Apr 2009 |
|
DE |
|
10 2008 027 784 |
|
Dec 2009 |
|
DE |
|
10 2012 108 158 |
|
Mar 2014 |
|
DE |
|
0 866 993 |
|
Sep 1998 |
|
EP |
|
2 587 295 |
|
May 2013 |
|
EP |
|
2000019099 |
|
Jan 2000 |
|
JP |
|
2013-097380 |
|
May 2013 |
|
JP |
|
WO 2004/053558 |
|
Jun 2004 |
|
WO |
|
WO 2012/110488 |
|
Aug 2012 |
|
WO |
|
WO 2012/122027 |
|
Sep 2012 |
|
WO |
|
Other References
Notification of Transmittal of Translation of the International
Preliminary Report on Patentability dated Jan. 12, 2016. cited by
applicant .
Reynaud E. G. et al., "Light sheet-based fluorescence microscopy:
more dimensions,more photons, and less photodamage", H F S P
Journal: Fontiers of Interdisciplinary Research in the Life
Sciences, International Human Frontier Science Program
Organization, FR, Bd. 2, Nr. 5, Oct. 1, 2008 (Oct. 1, 2008), Seiten
266-275, XP008129341, ISSN: 1955-2068. cited by applicant .
N. Jahrling et al., "Ultramicroscopy a a novel light sheet based
imaging technique created by various research disciplines;
Ultramikroskopie a eine neue bildgebende Technotogie auf Basis von
Laser Light-Sheets, entwickelt in Zusammerarbeit mehrerer
Forschungsdisziplinen", E & I Elektrotechnik Und
Informationstechnik, Springer-Verlag, Vienna, Bd. 128, Nr. 10, Oct.
1, 2011 (Oct. 1, 2011), Seiten 352-358, XP019983407, ISSN:
1613-7620. cited by applicant .
"Selective Plane Illumination Microscopy Techniques in
Developmental Biology" by J. Huisken et al., published in 2009 in
the journal Development, vol. 136, p. 1963. cited by applicant
.
"Optical thin-film materials with low refractive index for
broadband elimination of Fresnel reflection", by J.-Q. Xi et al.,
Published in 2007 in Nature Photonics, vol. 1, pp. 176-179. cited
by applicant .
International Search Report and Written Opinion for Application No.
PCT/EP2014/064550 dated Oct. 13, 2014. cited by applicant .
German Search Report dated Jul. 1, 2014. cited by applicant .
Emmauel G. Reynaud et al. , "Light sheet-based fluorescence
microscopy: more dimensions, more photons, and less photodamage",
HFSP Journal, Oct. 2008, vol. 2, No. 5, p. 266-275. cited by
applicant.
|
Primary Examiner: Pham; Thomas K
Assistant Examiner: Diedhiou; Ibrahima
Attorney, Agent or Firm: Haug Partners LLP
Claims
The invention claimed is:
1. An arrangement for light sheet microscopy comprising: a sample
vessel for receiving a sample that is located in a medium, the
sample vessel having a covering and being oriented with respect to
a planar reference surface; illumination optics with an
illumination objective for illuminating the sample with a light
sheet; and detection optics with a detection objective; wherein an
illumination optical axis of the illumination objective and the
light sheet lies in a plane which forms a nonzero illumination
angle .beta. with a normal of the reference surface; wherein a
detection optical axis of the detection objective forms a nonzero
detection angle .delta. with the normal of the reference surface;
wherein at least one bulge, which is transparent to illumination
light and detection light, is formed at the covering; wherein the
bulge has an inner surface and an outer surface for receiving the
sample in the bulge; wherein a shape of the at least one bulge, a
position of the bulge during observation, and a location of the
illumination and detection optical axes are selected so that the
illumination and detection optical axes form a minimal angle of
from 0.degree. to 5.degree. with the normals of the inner and outer
surfaces of the bulge at least in a region where the illumination
and detection optical axes pass through the surfaces; wherein the
at least one bulge comprises: first plate-shaped element; and a
second plate-shaped element; wherein each of the plate-shaped
elements projects from the covering and from the sample wherein
each of the plate-shaped elements projects from the covering and
from the sample vessel; wherein each of the plate-shaped elements
have parallel, planar inner and outer surfaces; wherein, at an
inner place on the bulge located at a greatest distance from the
rest of the sample vessel, the inner surface of the first
plate-shaped element and the inner surface of the second
plate-shaped element make contact at at least one point; wherein,
at an outer place on the bulge located at a greatest distance from
the rest of the sample vessel, the outer surface of the first
plate-shaped element and the outer surface the second plate-shaped
element make contact at at least one point; wherein normals of the
inner and outer surfaces of the first plate-shaped element coincide
with the illumination optical axis of the illumination objective;
and wherein normals of the inner and outer surfaces of the second
plate-shaped element coincide with the detection optical axis of
the detection objective.
2. The arrangement for light sheet microscopy according to claim 1;
wherein the at least one bulge is channel-shaped or
pyramid-shaped.
3. The arrangement for light sheet microscopy according to claim 1;
wherein the inner surface of the at least one bulge is
functionalized for the growth of cells.
4. The arrangement for light sheet microscopy according to claim 1;
wherein: the illumination objective and detection objective are
arranged below the sample vessel; the covering is formed as vessel
bottom; and the at least one bulge is formed as a depression.
5. The arrangement for light sheet microscopy according to claim 4;
wherein the sample vessel is formed as microtiter plate with a
plurality of bulges formed as wells, and a pyramid-shaped
depression is formed at each well.
6. The arrangement for light sheet microscopy according to claim 4;
wherein the depression is filled with a gel or alginate.
7. The arrangement for light sheet microscopy according to claim 1;
wherein: the illumination objective and detection objective are
arranged above the sample vessel; the covering is formed as a
vessel cover; and the at least one bulge is formed as a
protuberance; and wherein the arrangement further comprises a means
for positioning the sample in an upper region of the sample vessel
or the protuberance with respect to a depth, the means being
arranged in the sample vessel within a working distance of the
illumination objective and detection objective.
8. The arrangement for light sheet microscopy according to claim 7;
wherein the sample vessel is formed as microtiter plate with a
plurality of pyramid-shaped protuberances in the vessel cover.
9. The arrangement for light sheet microscopy according to claim 8;
wherein the microtiter plate is formed so as to be rotatable.
10. The arrangement for light sheet microscopy according to claim
7; wherein the means for positioning the sample in the upper region
of the sample vessel or the protuberance comprises a membrane that
is permeable to nutrient solutions, a platform with a plurality of
openings, or a strip.
11. The arrangement for light sheet microscopy according to claim
10; wherein the membrane, the platform, or the strip is made of
gel.
12. The arrangement for light sheet microscopy according to claim
1; wherein the illumination optics, the detection optics, or both
include a corrective means for reducing aberrations resulting from
the oblique passage of illumination light and/or light to be
detected through the surfaces.
13. The arrangement for light sheet microscopy according to claim
12; wherein the corrective means includes corrective lenses in the
illumination objective and/or in the detection objective.
14. The arrangement for light sheet microscopy according to claim
1; wherein the vessel bottom and/or the vessel cover are/is made of
a material having a refractive index that differs by less than 5%
from a refractive index of the medium in which the sample is
located.
15. The arrangement for light sheet microscopy according to claim
14; wherein the material comprises a nanostructured mix material
comprising: a first component; and a second component; wherein a
refractive index of the first component is less than the refractive
index of the medium, and a refractive index of the second component
is greater than the refractive index of the medium; and wherein
mean structure sizes of regions of material of the first component
have a mean diameter that is less than the light wavelengths of the
light which is to be used for illumination and which is to be
detected.
16. The arrangement for light sheet microscopy according to claim
1; wherein a sum of the illumination angle .beta. and detection
angle .delta. is 90.degree..
17. The arrangement for light sheet microscopy according to claim
13; wherein the corrective lenses comprise cylindrical lenses,
tilted lenses, or lenses that are not arranged axially.
18. The arrangement for light sheet microscopy according to claim
12; wherein the corrective means includes: corrective elements with
aspherical surfaces or with free-form surfaces; or adaptive optical
elements arranged in the illumination beam path, in the detection
beam path, or in both for manipulating phase fronts of the
illumination light and/or detection light; or a combination
thereof.
19. The arrangement for light sheet microscopy according to claim
18; wherein the corrective means includes the adaptive optical
elements; and wherein the adaptive optical elements comprise
deformable mirrors, spatial light modulators, phase plates, or a
combination thereof.
Description
The present application claims priority from PCT Patent Application
No. PCT/EP2014/064550 filed on Jul. 8, 2014, which claims priority
from German Patent Application No. DE 10 2013 107 298.4 filed on
Jul. 10, 2013, the disclosures of which are incorporated herein by
reference in their entirety.
FIELD OF THE INVENTION
It is noted that citation or identification of any document in this
application is not an admission that such document is available as
prior art to the present invention.
The invention is directed to an arrangement for light sheet
microscopy. An arrangement of this kind includes a sample vessel
for receiving a sample that is located in a medium, this sample
vessel being oriented with respect to a planar, usually horizontal,
reference surface. The arrangement further includes illumination
optics with an illumination objective for illuminating the sample
with a light sheet, and the optical axis of the illumination
objective and the light sheet lie in a plane which forms an
illumination angle .beta. not equal to zero with the normal of the
reference surface. Finally, the arrangement for light sheet
microscopy also comprises detection optics with a detection
objective having an optical axis which forms a detection .delta.
not equal to zero with the normal of the reference surface. The
illumination objective and detection objective can also be
configured as a so-called double-objective such as is described,
for example, in EP 0 866 993 B1. In this case, the two objectives
are put together in a shared constructional unit, and the
respective optics--i.e., objectives with associated beam paths and
optical elements arranged therein share some elements.
An arrangement of this type is used particularly in the examination
of biological samples in which the sample is illuminated by a light
sheet, the plane of which intersects the optical axis of detection
at an angle not equal to zero. The light sheet typically forms a
right angle with the detection direction which generally
corresponds to the optical axis of the detection objective. Spatial
recordings of even thick samples can be made relatively quickly
with this technique, also referred to as SPIM (selective plane
illumination microscopy). A graphic, spatially extensive
representation of the sample is made possible based on optical
sections combined with a relative movement in a direction
perpendicular to the section plane.
The SPIM technique is preferably used in fluorescence microscopy,
where it is accordingly also referred to as LSFM (light sheet
fluorescence microscopy). The LSFM technique has a number of
advantages over other established methods such as confocal laser
scanning microscopy or two-photon microscopy. Since widefield
detection can be carried out, larger sample regions can be
acquired. Although the resolution is somewhat lower than in
confocal laser scanning microscopy, the LSFM technique can be used
to analyze thicker samples because the penetration depth is
greater. Further, this method has the least light stress on the
samples, which, among other things, reduces the risk of
photobleaching of a sample because the sample is only illuminated
by a thin light sheet at an angle to the detection direction not
equal to zero.
Instead of using a purely static light sheet, a quasistatic light
sheet can also be generated through fast scanning of the sample
with a light beam. The light sheet-type illumination is brought
about in that the light beam undergoes a very fast relative
movement with respect to the sample to be observed and is thus
strung together over and over in a temporally consecutive manner.
The integration time of the camera on whose sensor the sample is
ultimately imaged is selected such that the scanning is concluded
within the integration time. Instead of a camera with a
two-dimensional array, a line sensor combined with a renewed
scanning (rescan) can also be used in the detection optics.
Further, confocal detection can also be carried out.
The SPIM technique has been described many times in the literature,
for example, in DE 102 57 423 A1 and in WO 2004/053558 A1 which is
based on the latter, and in the survey article "Selective Plane
Illumination Microscopy Techniques in Developmental Biology" by J.
Huisken et al. published in 2009 in the journal Development, vol.
136, p. 1963.
One of the chief applications of light sheet microscopy is for
imaging intermediate-sized organisms having a size of some hundreds
of micrometers to a few millimeters. Generally, these organisms are
embedded in an agarose gel which is located in turn in a glass
capillary. The glass capillary is inserted from above or below into
a sample chamber filled with water, and the sample is pushed some
distance out of the capillary. The sample in the agarose is
illuminated by a light sheet and the fluorescence is imaged on a
camera by a detection objective oriented perpendicular to the light
sheet and, therefore, also perpendicular to the light sheet
optics.
This method of light sheet microscopy has three sizable
disadvantages. For one, the samples to be examined are relatively
large and derive from developmental biology. Further, because of
the sample preparation and the dimensions of the sample chamber,
the light sheet is relatively thick and accordingly limits the
attainable axial resolution. In addition, the sample preparation is
complicated and is not compatible with standardized sample
preparations or standardized sample holders such as are
conventionally used in fluorescence microscopy for individual
cells.
In order to circumvent these limitations at least partially, a SPIM
construction was recently developed in which the illumination
objective and the detection objective are perpendicular to one
another and are directed onto the sample from above at an angle of
45.degree. in each instance. When, for example, the specimen stage
on which the sample holder is fixed or some other horizontal plane
is used as reference surface, the illumination angle .beta. and the
detection angle .delta. are both 45.degree.. A construction of this
kind is described, for example, in WO 2012/110488A2 and
WO2012/122027A2.
In a construction such as this, the sample is located, for example,
on the bottom of a petri dish. The petri dish is filled with water,
and the illumination objective and detection objective are immersed
in the liquid which also takes on the function of an immersion
medium. This approach offers the advantage of higher resolution in
axial direction, since a thinner light sheet can be generated.
Smaller samples can then also be examined owing to the higher
resolution. Sample preparation is also made significantly easier.
The great drawback still consists in that the sample preparation
and sample holder still do not correspond to the standard mentioned
above. Accordingly, the petri dish must be relatively large so that
the two objectives can be immersed in the dish without hitting the
edge of the dish. Microtiter plates--also known as multi-well
plates--which are standard in many branches of biology and in
fluorescence microscopy analysis of individual cells cannot be used
with this method because the objectives cannot be immersed in the
very small wells of the plate. Further, this method has the
disadvantage that it is not readily possible to analyze a large
number of samples in a short period of time (high-throughput
screening) because the objectives must be cleaned when changing
samples in order to avoid contaminating the different samples.
SUMMARY OF THE INVENTION
It is the object of the invention to further develop an arrangement
for light sheet microscopy of the type described in the
introduction such that high-throughput analysis of samples is
facilitated in that the use of microtiter plates, i.e., sample
holders which can receive a large number of samples, is
facilitated.
This object is met for an arrangement for light sheet microscopy of
the type described in the introduction in that at least one bulge
which is at least partially transparent to illumination light and
detection light is formed at the covering for receiving the sample,
this bulge having an inner interface and an outer interface. This
substantially facilitates access of the objectives to the sample.
In particular, microtiter plates as well as rotatable microtiter
plates--can be used, and the wells of these microtiter plates can
then be configured with smaller lateral dimensions than if the
sample were located at the vessel bottom, particularly when an
upright microscope configuration is used for the analysis.
In this respect, it is key that the shape of the bulge, the
position of the bulge during observation, and the location of the
optical axes of the illumination objective and detection objective
are adapted to one another in order to prevent or minimize
aberrations which would result when a beam path extends obliquely
through the interfaces and, therefore, when light enters and exits
the sample vessel obliquely. The adaptation is carried out in such
a way that the optical axes of the illumination objective and
detection objective form a minimal angle with the normals of the
inner interface and outer interface at least in the region where
the optical axes pass through the interfaces, i.e., an angle which
is equal to zero or only deviates from zero by a few degrees,
approximately up to 5.degree.. If the optical axes and the
interfaces are perpendicular to one another, only spherical
aberrations occur which can be corrected as in known microscope
objectives adapted to coverslips.
In principle, the bulge can have any shape provided that the
above-mentioned condition is met. For example, the bulge can have a
half-barrel shape or half-sphere shape, in which case the optical
axes of the two objectives coincide with normals to tangents at the
surface of the half-barrel in the best possible configuration of
the adapted arrangement.
In a particularly preferred embodiment, the at least one bulge
comprises two plate-shaped elements which project from the covering
and sample vessel and which have parallel interfaces which, at that
place on the bulge located at the greatest distance from the rest
of the sample vessel--which, in case the bulge is formed as a
depression, is the lowest point of the depression and, in case the
bulge is formed as a protuberance, is the highest point of the
protuberance--make contact at at least one point and, at this
point, the depression or the protuberance and the sample vessel or
the vessel cover, respectively, terminate at the bottom or at the
top. In the region where the optical axis of the illumination
objective passes through, the normals of the interfaces of a first
plate-shaped element coincide with this optical axis such that the
normals and the optical axis are parallel to the optical axis of
the illumination objective at every location on the interfaces of
the plate-shaped element. Correspondingly, the normals of the
interfaces of a second plate-shaped element coincide with the
optical axis of the detection objective, i.e., are parallel to this
optical axis at every location on the interfaces of the second
plate-shaped element. This allows a greater flexibility for
adapting with respect to the position of the bulge in relation to
the two objectives. However, the plate shape which implies a
parallel attitude of the inner interface and outer interface with
respect to one another is not compulsory and, particularly in the
region where the two plate-shaped elements make contact, this
region can be provided with a small curvature on the inner side
such that, on the one hand, e.g., in case of a depression, the
depression is strengthened at its lowest point and, on the other
hand, a stubborn adherence of impurities which would occur when two
plane plates abut at an angle, i.e., in the case of a depression
with an at least partially V-shaped cross section, is also
prevented.
The sum of the illumination angle .beta. and detection angle
.delta. is preferably 90.degree., which facilitates the arrangement
of a detector in the beam path. At other angles, it must be ensured
that the image plane, i.e., the plane in which the detector is
situated, intersects the object plane, i.e., the plane irradiated
by the light sheet, and the object-side principal plane of the
detection objective in a straight line.
The at least one bulge can be channel-shaped, and a plurality of
channels can be arranged one behind the other in the sample vessel,
for example, in the vessel bottom. In a particularly preferred
embodiment, the bulge is pyramid-shaped so that the two
plate-shaped elements have a triangular shape and are supplemented
by two further plate-shaped elements. This makes it possible to
analyze a sample located in the bulge from four different sides,
which can be advantageous when the sample attaches to one side. In
addition, pyramid-shaped bulges can be arranged in grid shape at
the vessel bottom or in the vessel cover such that the sample
vessel can also be configured as a microtiter plate with a
plurality of pyramid-shaped bulges of this kind. The channel-shaped
configuration can also be utilized for a microtiter plate when the
individual channels are divided into individual sections by
partitioning elements such as crosspieces.
A sample vessel with a depression of the type mentioned above can
be made of glass but, in a more economical variant, can also be
made of plastic, e.g., by means of a deep drawing process, when the
depression is channel-shaped.
At least a portion of the inner interface of the bulge is advisably
functionalized for growing cells on this interface, i.e., it is
coated with a special structure to which the surface structures of
the cells adhere and are anchored. For individual cells which are
to be analyzed in high throughput with the present light sheet
microscope arrangement, the growth conditions can be even better
adapted to the natural growth environment in that the at least one
bulge, i.e., for example, a channel or pyramid-shaped depression,
is filled with a gel or alginate by which a spatial matrix can be
simulated.
As has already been indicated, in a preferred embodiment in which
the illumination objective and detection objective are arranged
below the sample vessel in an inverted configuration, the covering
is formed as vessel bottom and the bulge is formed as depression in
the vessel bottom. In this way, the access of the objective to the
sample is substantially facilitated. In particular, microtiter
plates with a plurality of wells can also be used, and a depression
for a sample is formed in each of these wells. The use of a
depression or depressions for examining many cell samples makes it
possible to increase the quantity of wells in a sample vessel
because the lateral dimensions--in the plane of the reference
surface--can be reduced.
In an upright configuration of the light sheet microscope, i.e.,
for observing from above, the vessel cover instead of the vessel
bottom can also be adapted as covering in a manner analogous to
that described for the vessel bottom, and the analysis of cells can
be carried out with an upright light sheet microscopy arrangement.
Therefore, in this embodiment of the invention, the illumination
objective and detection objective are arranged above the sample
vessel. In this case, at least one protuberance is formed in the
vessel cover instead of a depression. The shape of the
protuberance, the position of the protuberance during observation,
and the location of the optical axes of the illumination objective
and detection objective are then likewise adapted to one another in
that the optical axes of the illumination objective and detection
objective form a minimal angle with the normals of the interfaces
at least in the region where the optical axes pass through the
interfaces. Like the depressions, the protuberances can be
channel-shaped, pyramid-shaped, half-barrel-shaped or
half-sphere-shaped.
In view of the fact that in microtiter plates the sample usually
sinks owing to gravity or settles at the lowest point, observation
is not readily possible when the illumination objective and
detection objective are arranged above the sample vessel. For this
reason, when the protuberances are formed in the vessel cover,
additional means for positioning the sample in the upper region of
the sample vessel with respect to the depth thereof are arranged in
the sample vessel within the working distance of the objectives.
The means for positioning can also be arranged in the vessel cover,
also within the working distance of the objectives in a
corresponding manner. The working distance for typical objectives
with a high numerical aperture usually ranges from a few hundred
micrometers to several millimeters.
Depressions or protuberances can be formed of plate-shaped
elements, and the inner interfaces can be functionalized. A further
possibility for configuring the sample vessel consists in using a
rotatable microtiter plate in which, first of all, the
protuberances in the cover face downward. The sample is placed in
this protuberance, which corresponds to a depression in the
charging position, and is fixed therein by the means for
positioning the sample in the at least one protuberance, for
example, by means of a plunger. Subsequently, the bottom is placed
on the rotatable microtiter plate from above and the microtiter
plate is closed. For purposes of analysis, this microtiter plate is
then rotated when it is to be used with an upright light sheet
microscope. Rotation is not required when it is to be used with an
inverted light sheet microscope.
The means for positioning the sample in the top one fourth or in
the at least one protuberance in the vessel cover advisably
comprise a membrane which is permeable to nutrient solutions, a
platform with a plurality of openings, or a strip. It is important
that the sample makes contact with the nutrient solution in every
case, but it may not sink into this nutrient solution due to
gravity. The membrane, the platform or the strip can also be made
of gel.
Due to the fact that the light passes through three different media
or two interfaces between each objective and the sample, spherical
aberrations also occur with vertical orientation of the objectives
with respect to the interfaces. Knowing the material for the bulge
in the shape of a depression in the vessel bottom or in the shape
of a protuberance in the vessel cover and knowing the thickness at
least of plate-shaped elements with parallel interfaces, these
aberrations can be corrected in the manner commonly implemented for
microscope objectives. In some cases, they are corrected with
respect to a specified coverslip thickness of a specified material.
Corrections of this type are particularly preferably carried out in
the detection objective, which generally has a higher numerical
aperture than the illumination objective.
In a preferred embodiment, the illumination optics and/or detection
optics include corrective means for reducing not only the
above-mentioned aberrations, but also aberrations resulting from
the passage of illumination light and/or light to be detected
through the interfaces at an angle other than 90.degree..
Therefore, special corrective lenses are preferably arranged in the
illumination objective and/or in the detection objective. In case
the objectives form an angle not equal to zero with the normals of
the interfaces, these corrective lenses can also include
cylindrical lenses, tilted lenses or lenses which are not arranged
on the optical axes. Corrective elements with aspherical surfaces
or free-form surfaces can also be utilized for correction.
Alternatively or in addition, corrective means in the form of
adaptive optical elements can be arranged in the illumination beam
path for manipulating the phase fronts of the illumination light
and/or detection light. In this connection, deformable mirrors,
spatial light modulators or phase plates are preferably used.
Another method for reducing aberrations is to use specially adapted
materials for the covering or for the protuberances in the vessel
cover or depressions in the vessel bottom.
In a particularly preferred embodiment, materials having a
refractive index that differs by less than 5% from the refractive
index of the medium in which the sample is located are used as
material for the depression or protuberance in the vessel bottom or
vessel cover, respectively. For example, when water, which has a
refractive index n.sub.d=1.33 at a wavelength .lamda..sub.d=578.56
nm, is used as medium in which the sample is located, examples of
suitable material for the covering are PTFE
(polytetrafluoroethylene, n.sub.d=1.35), CYTOP.RTM. (n.sub.d=1.34)
or PFEP (fluorinated ethylene propylene, n.sub.d=1.34).
Perfluorodioxolane polymers which likewise have a refractive index
generally between 1.33 and 1.36 can also be used. Teflon.RTM. AF
which usually has a refractive index n.sub.d=1.32 is also a
particularly well-suited material. This material is an amorphous
polymer. In this case, the glass transition temperature can be
adjusted in such a way that the polymer in cooled state has the
refractive index of the medium in which the sample is located.
Other amorphous polymers having an adjustable glass transition
temperature can also be used, of course.
If the refractive indices do not coincide exactly, aberrations
continue to occur, although to a lesser degree. Therefore, in order
to further prevent these aberrations, the bulge should be as thin
as possible and should be no thicker than some hundreds of
micrometers. If the covering serves simultaneously as the bottom of
the sample vessel, as is the case with an inverted arrangement, a
sufficient stability must, of course, be ensured with respect to
the pressure exerted by the medium in which the sample is located.
This is not necessary when the covering serves as cover of the
sample vessel for upright observation. In this case, the material
can be shaped in a substantially thinner manner with thicknesses of
less than 100 .mu.m.
In a further step which is easily implemented particularly in
upright light sheet microscopy, immersion objectives can again be
used. If the same medium is used both as immersion medium and as
medium for receiving the sample, i.e., water, for example, and the
refractive index of water, and if a material is used for the
protuberances or depressions in the vessel cover or vessel bottom
that has a refractive index almost identical to that of water,
there is no noticeable scatter or refraction on the interfaces, and
the objectives need not be corrected further.
A nanostructured mix material composed of a first component and a
second component can also be used as material for the protuberances
and depressions in the vessel cover and in the vessel bottom. The
refractive index of the first component is less than the refractive
index of the medium for receiving the sample, and the refractive
index of the second component is greater than the refractive index
of the medium for receiving the sample. If the mean structure size
of the material of the first component is less than the light
wavelengths of the light which is to be used for illumination and
which is to be detected, there results an effective refractive
index for the mix material which can likewise be adapted to the
refractive index of the medium depending on the size of the regions
and quantity of regions so that the refractive index is in the
range of 5% around the refractive index of the medium for embedding
the sample. For example, nanoporous silicon dioxide can be used. In
this case, the first component is air and the second component is
silicon dioxide. These kinds of nanostructured materials are
described in connection with the production of antireflective
layers, for example, in the article "Optical thin-film materials
with low refractive index for broadband elimination of Fresnel
reflection", by J.-Q. Xi et al., published in 2007 in Nature
Photonics, Vol. 1, pages 176-179.
It will be appreciated that the features mentioned above and those
to be described hereinafter can be used not only in the indicated
combinations but also in other combinations or individually without
departing from the scope of the present invention.
The invention will be described more fully in the following by way
of example with reference to the accompanying drawings which also
disclose key features of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an arrangement for light sheet microscopy;
FIG. 2 shows an example of a sample vessel with depressions;
FIG. 3 shows an example of a vessel cover or vessel bottom;
FIGS. 4 a)-c) show various possibilities for arranging a sample in
the upper region of a well of a microtiter plate; and
FIG. 5 shows the use of a rotatable microtiter plate.
DETAILED DESCRIPTION OF EMBODIMENTS
It is to be understood that the figures and descriptions of the
present invention have been simplified to illustrate elements that
are relevant for a clear understanding of the present invention,
while eliminating, for purposes of clarity, many other elements
which are conventional in this art. Those of ordinary skill in the
art will recognize that other elements are desirable for
implementing the present invention. However, because such elements
are well known in the art, and because they do not facilitate a
better understanding of the present invention, a discussion of such
elements is not provided herein.
The present invention will now be described in detail on the basis
of exemplary embodiments.
First, FIG. 1 shows the basic construction of an arrangement for
light sheet microscopy which allows easy access to the sample to be
examined and therefore meets the prerequisite for use in
high-throughput analysis of individual cells. The arrangement is
configured in the present instance as an inverted light sheet
microscope, but can easily be transferred to an upright light sheet
microscope. A sample 3 is located in a medium 2 in a sample vessel
1. The sample vessel 1 is oriented with respect to a planar
reference surface which is defined in this case by the horizontal
surface of a specimen stage 4. The arrangement further includes
illumination optics with a light source 5 and an illumination
objective 6 for illuminating the sample 3 with a light sheet. The
light sheet and the optical axis 7 of the illumination objective 6
lie in a plane which forms an illumination angle .beta. not equal
to zero with the normals of the reference surface. Light coming
from the sample is imaged on a detector 10 via detection optics
having a detection objective 8 with optical axis 9 forming a
detection angle .delta. not equal to zero with the normals of the
reference surface. The detector 10 transforms the registered
intensity into image data which can be further processed. The
illumination angle .beta. and detection angle .delta. are identical
in the present case, but this is not compulsory. For example, when
the apertures of the two objectives differ, the angles can also be
adjusted differently based on the space requirement.
The illumination objective 6 and detection objective 8 are arranged
below the sample vessel 1. The sample vessel 1 has a vessel bottom
11 which is transparent to illumination light and detection light
and which has an inner interface 12 and an outer interface 13. At
least one depression 14 which is transparent to illumination light
and detection light is formed at the vessel bottom 11 for
depositing the sample 3 into the depression. In this connection, it
is sufficient when the sample vessel 1 is transparent in the region
of the depression 14, but it is generally simpler to produce it
from a uniform material such as glass or deep-drawn plastic. By
depositing the sample 3 in this depression 14, the sample 3 is more
easily accessible to the optical arrangement of the light sheet
microscope, the illumination objective 6 and the detection
objective 8. A sample vessel 1 with a plurality of depressions 14
of this type is better suited for a high-throughput analysis of
individual cells than a vessel with a flat bottom, since the
individual wells in a multi-well plate or microtiter plate of this
kind can have smaller lateral dimensions because the sample is
deposited in the depression. Therefore, the microtiter plates need
not be changed as often.
The shape of the depression 14, the position of the depression 14
during observation, and the locations of the optical axes 7 and 9
of the illumination objective 6 and detection objective 8 are
adapted to one another in that these optical axes 7, 9 of the
illumination objective 6 and detection objective 8 form a minimal
angle with the normals of the inner interface 12 and outer
interface 13 at least in the region where optical axes 7 and 9 pass
through interfaces 12 and 13. The occurrence of aberrations
resulting from light impinging on and exiting obliquely through the
interfaces can be minimized in this way. The angle is preferably
zero.
In the example shown in FIG. 1, the at least one depression 14 has
a first plate-shaped element 15 and a second plate-shaped element
16 projecting from the vessel bottom 11. The inner interface 12 is
arranged parallel to the outer interface 13 in each of the
plate-shaped elements 15 and 16. At the lowest point of the
depression 14, the two plate-shaped elements 15 and 16 contact at
at least one point, the normals of the interfaces 12, 13 of the
first plate-shaped element 15 coincide with the optical axis 7 of
the illumination objective 6, and the normals of the interfaces 12,
13 of the second plate-shaped element 16 coincide with the optical
axis 9 of the detection objective 8. The sum of the illumination
angle .beta. and detection angle .delta. is 90.degree. in the
present instance, but can also deviate from this. This arrangement
has the great advantage that aberrations such as can occur when
light passes obliquely through the interfaces 12, 13 can be
entirely prevented. Further corrections of the illumination
objective 6, which generally has a small numerical aperture on the
order of 0.3, are no longer necessary because the light sheet to be
generated should be as thin as possible. However, further
corrections are advantageous for the detection objective 8 which
generally has a high numerical aperture on the order of 1.0 and in
certain cases also for the illumination objective. The corrective
means can comprise, for example, corrective lenses in the
illumination objective 6 or in the detection objective 8, or
adaptive optical elements for manipulating the phase fronts of the
illumination light and/or detection light which are arranged in the
illumination beam path or in the detection beam path and are
preferably configured as deformable mirrors, spatial light
modulators or phase plates.
To completely eliminate scattering and refraction at interfaces 12,
13, the vessel bottom 11 can also be shaped from a material which
has a refractive index which differs by less than 5% from the
refractive index of the medium 2 in which the sample 3 is located.
Amorphous polymers having glass transition temperatures which can
be adjusted such that, when cooled, the material has exactly the
required refractive index are particularly suitable for this
purpose. A nanostructured mix material formed, for example, from
nanoporous silicon dioxide, i.e., silicon dioxide with a plurality
of cylindrical holes, can also be used as material for the vessel
bottom 11. In every case, the vessel bottom 11 should be as thin as
possible in order to suppress aberrations as far as possible. The
example shown in FIG. 1 can also be transferred in an equivalent
manner to an upright arrangement of the illumination objective 6
and detection objective 8, in which case, instead of a depression
14 in the vessel bottom 11, the vessel cover has a corresponding
protuberance.
FIG. 2 shows an example for a sample vessel 1 which is suitable for
high-throughput analysis of cells. Two channel-shaped depression 14
arranged parallel to one another are shown in a section of a sample
vessel 1. Each of these depressions 14 is divided by crosspieces 17
into individual wells which make it possible to arrange a plurality
of samples next to one another in a depression 14 without the
possibility of mutual contamination.
Instead of the vessel bottom 11, a corresponding vessel cover 18
can also be configured in this way. FIG. 3 shows a section from a
vessel cover 11 on which are arranged a plurality of pyramid-shaped
protuberances 19, each of which covers a well in the sample vessel
1. In an equivalent manner, the vessel bottom 1 can also be
configured in this way.
The outer interfaces 13 in the depressions 14 or protuberances 19
can be functionalized for growing cells on this interface such
that, for example, cells can also attach to the protuberances 19
without additional aid. The depression 14 or the protuberance 19
can also be filled with a gel or alginate to immobilize the
sample.
To facilitate observation of the samples and to allow the wells of
a microtiter plate to be designed with the smallest possible
lateral diameter, sample vessels of this type which are provided
for upright observation preferably have means for positioning the
sample in the upper region of the sample vessel 1 with respect to
depth within the working distance of the illumination objective and
detection objective or for corresponding positioning within the
working distance in the protuberance 19 in the vessel cover 18.
These means are shown in FIGS. 4a)-c). The box-shaped element
represents a well 20 in a multi-well plate in a sample vessel 1 for
upright observation. In FIG. 4 a), a permeable membrane 21 is
arranged in the upper region, the sample 3 being supported thereon.
This permeable membrane 21 ensures contact with a comparatively
large volume of nutrient fluid to enable the growth of the cells.
The membrane 21 allows diffusion of nutrients and also supports the
sample 3. A platform 22, which is flat, for example, and which has
openings as is shown in FIG. 4 b) can also be used instead of a
membrane 21. The platform can be made of glass, for example, so
that the sample preparation can proceed substantially in accordance
with standard protocols. The cell culture can also be immobilized
in a matrix gel. In FIG. 4 a), a protuberance 19 of a vessel cover
18 in the form of a channel or section of a channel is shown on the
well 20. The use of a flat covering, for example, a sheet 23, as is
shown in FIG. 4 b), is also possible in principle. The sheet 23 can
be glued or welded to the sample vessel. A further configuration is
shown in FIG. 4 c) which illustrates a strip 24 projecting into the
center of the well. In this instance, the protuberance 19 has a
half-barrel shape. The supporting elements mentioned above,
membrane 21, platform 22 and strip 24, can also be made of gel
provided it possesses sufficient rigidity.
The use of rotatable microtiter plates as is shown in FIG. 5 is
also conceivable. In this case, the sample 3 is first placed in a
well 20 of the microtiter plate. The well 20 is funnel-shaped in
the present instance by way of example. The well 20 is filled with
the medium 2. Subsequently, a funnel-shaped element 25 having a
membrane 21 at its smaller-diameter end is inserted into the
funnel-shaped well 20. The microtiter plate is then closed by the
vessel bottom 11. Subsequently, the plate is rotated, and the
sample can then be observed with an upright arrangement for light
sheet microscopy.
While this invention has been described in conjunction with the
specific embodiments outlined above, it is evident that many
alternatives, modifications, and variations will be apparent to
those skilled in the art. Accordingly, the preferred embodiments of
the invention as set forth above are intended to be illustrative,
not limiting. Various changes may be made without departing from
the spirit and scope of the inventions as defined in the following
claim.
LIST OF REFERENCE NUMERALS
1 sample vessel 2 medium 3 sample 4 specimen stage 5 light source 6
illumination objective 7 optical axis 8 detection objective 9
optical axis 10 detector 11 vessel bottom 12 inner interface 13
outer interface 14 depression 15 first plate-shaped element 16
second plate-shaped element 17 crosspiece 18 vessel cover 19
protuberance 20 well 21 membrane 22 platform 23 sheet 24 strip 25
funnel-shaped element
* * * * *